Butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8, also known as pseudocholinesterase) is an enzyme found in the plasma, among other tissues, of all vertebrates in which it has been sought (Silver, A. The Biology of Cholinesterase, North-Holland, Amsterdam, 1974). The existence of this enzyme in human plasma was formally demonstrated 50 years ago (Alles, G. A. and Hawes, R. C., J. Biol. Chem., 133:375, 1940), but its normal physiological role remains unknown. However, butyrylcholinesterase is responsible for the hydrolysis and inactivation of muscle relaxants such as succinylcholine and related anaesthetics (LaDu, B. M., Ann. N.Y. Acad. Sci., 179:648, 1971), substances currently in clinical use. Butyrylcholinesterase is also responsible for degrading the majority of the cocaine ingested by a drug abuser (Stewart, D. J. et al., Life Scie., 20:1557, 1977; Jatlow, P., et al., Anesth. Anag., 58:235, 1979; Stewart, D. J. et al., Clin. Pharmacol. Ther. 25:464, 1979).
The gene for human butyrylcholinesterase exists as a "wild-type" (normal) allele and several defective alleles which are present in as much as 5% of the population (reviewed in Whittaker, M., Anaesthesia, 35:174, 1980; Evans, R. T., CRC Crit. Rev. Clin. Lab. Sci., 23:35, 1985). In approximately 1 in 2800 individuals, their genotype results in a severe deficiency in butyrylcholinesterase. When these individuals are treated with succinylcholine during the induction of general anaesthesia prior to surgery, the resulting paralysis is greatly prolonged compared to the normal population. During this period the patient is unable to breathe, a condition known as apnea, and must be artificially ventilated until the succinylcholine is degraded by secondary mechanisms. This is considered to be a potentially life-threatening situation. Butyrylcholinesterase activity may also be reduced sufficiently from normal levels to induce succinylcholine sensitivity during pregnancy (Wildsmith, J. A. W., Anaesthesia, 27:90, 1972; Weissman, D. B. and Ehrenwerth, J., Anesth. Analg., 62:444, 1983), by certain diseases such as hepatitis (Singh, D. C. et al., J. Ind. Med. Assoc.. 66:49, 1976) or as a consequence of various medications (Foldes, F. F., Enzymes in Anaesthesiology, Springer-Verlag, N.Y., 1978).
Toxicologically, cocaine is also well tolerated by the majority of the population. Nevertheless there is a small incidence of sudden death related to acute cocaine abuse see Clouet, D. et al., Mechanisms of Cocaine Abuse and Toxicity, NIDA Research Monograph 88; Johanson, C. and Fischman, M. W., Pharmacol. Rev. 41:3, 1889). The physiological basis for this difference in susceptibility is not known. However, it has been argued that a deficiency in butyrylcholinesterase could contribute to an individual's sensitivity (Stewart, D. J. et al, supra, 1979; Jatlow, P., (supra, 1979; Anton, A. H., Drug Intell. Clin. Pharm., 22:914, 1988; Devenyl, P., Ann. Int. Med.. 110:167, 1989).
A number of compounds of the organonhosphate type are used as pesticides (e.g. malathion) or neurotoxic chemical warfare agents (e.g. soman; Silver, A., supra, 1974; Aldridge, W. N. and Reiner, E., Enzyme Inhibitors as Substrates, North-Holland, Amsterdam, 1972). These compounds exert their toxic effects by inhibiting acetylcholinesterase, an enzyme found on erythrocytes and at cholinergic synapses where it plays an essential role in proper neurological and neuromuscular function. Butyrylcholinesterase is also inhibited by these compounds because of the similarity of its active site to that of acetylcholinesterase (Soreq, H. and Prody, C. A., in: Computer Assisted Modeling of Receptor-Ligand Interactions, Alan R. Liss, N.Y., 1989). Therefore, plasma butyrylcholinesterase and erythrocyte acetylcholinesterase afford some protection to synaptic acetylcholinesterase from these neurotoxins since the toxins themselves are inactivated by the reactions that inhibit these enzymes. Only those toxin molecules that survive in the circulatory system without reacting with the plasma cholinesterases are capable of attacking synaptic acetylcholinesterase. It follows that an individual's susceptibility to these compounds is determined in part by the amount of cholinesterase present in the blood. It has been shown that administration of bovine serum acetylcholinesterase to mice increases their resistance to organophosphate poisoning (Rauch, L., Ashani, Y., Levy, D., de la Hoz, D., Wolfe, A. D. and Doctor, B. P., Biochem. Pharmacol., 38:529, 1989).
Butyrylcholinesterase is present in human plasma, serum or whole blood. Methods have been developed for obtaining butyrylcholinesterase from plasma. These can be classified in two groups: those in which the plasma is first fractionated by a precipitation method and those in which the plasma is chromatographed directly.
The earliest methods employed ethanol or ammonium sulfate as precipitants. Cohn et al. (J. Amer. Chem. Soc., 68:459, 1946) found that the majority of "plasma esterase" partitioned into one fraction, designated IV-4, during the fractionation of human plasma by ethanol. Subsequently, Surgenor and Ellis (J. Amer. Chem. Soc., 76:6049, 1954) extended this method by repetitive precipitations to produce human butyrylcholinesterase (designated fraction IV-6-4) of about 20% purity with a yield of 7%. An intermediate fraction (IV-6-3) obtained by this procedure was further purified by chromatography on hydroxylapatite and Dowex anion exchange resin (Malstrom et al., Acta Chem. Scand., 10:1077, 1956). While this last procedure produced butyrylcholinesterase of high (at least 80%) purity, the overall recovery was poor, no better than 3%.
Several other methods have been developed which employ ammonium sulfate precipitation as an early step. These procedures either produced crude enzyme (no more than 10% purity; Goedde, H. W. et al., Human Genet., 1:311, 1965) or incorporated preparative electrophoresis, a technique which is impractical for any large scale process, to achieve higher degrees of purity with about 10% yields (Svensmark, O. and Kristensen, P., Biochim. Biophys. Acta. 67:441, 1963; Haupt, H. et al., Blut, 14:65, 1966). Because of these drawbacks, these methods have been abandoned for any application requiring highly purified butyrylcholinesterase in large (commercial) quantities.
Present methods employ the chromatographic purification of butyrylcholinesterase from defibrinated plasma and are based on the ability of the enzyme to bind to conventional anion exchange resins under acidic (pH 4) conditions (Connell, G. E. and Shaw, R. W., Can. J. Biochem. Physiol., 39:1019, 1961). When optimized, anion exchange chromatography at pH 4 of human plasma achieves a 400- to 800-fold purification of butyrylcholinesterase (i.e. to a purity of 2-4%) in a single step (Das, P. K. and Liddell, J., Biochem. J., 116:875, 1970; Meunsch, H. et al., Eur. J. Biochem., 70:217, 1976). The subsequent steps used by these groups to further purify the enzyme were supplanted by affinity chromatography on procainamide-agarose (Lockridge, O. and LaDu, B. N., J. Biol. Chem., 253:361, 1978) which achieved a two-step purification of butyrylcholinesterase to 88% purity with a 70% yield. Further refinements of the method added an additional anion exchange step at pH 7 (Lockridge, O. and LaDu, B. N., J. Biol. Chem., 287:12012, 1982; Lockridge, O. et al., J. Biol. Chem., 262:549, 1987), producing virtually homogeneous enzyme with an overall yield of 30-40%.